Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival

Abstract

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.

Figure 1. Identification of CK1α as a CARMA1-binding partner

a, Schematic and sequence of CK1α. Peptides identified by a mass spectrometry analysis of CARMA1-containing complexes are highlighted in grey. b, Interaction between HA-CK1α and V5-CARMA1 in HEK293T cells by immunoprecipitation (IP) and immunoblot (IB). c–e, IP/IB as indicated, in Jurkat T lymphocytes stimulated with 1 μg/ml anti-CD3 and anti-CD28 (c–d), and in BJAB B cells stimulated with 20 ng/ml PMA and 300 ng/ml ionomycin (e). Filled and open symbols, non phosphorylated and phosphorylated forms. Ub, ubiquitin. f, Confocal images of CD3 and CK1α following CD3 crosslinking in Jurkat. Nuclei counterstaining is shown in blue. g, IP/IB of V5- CARMA1 binding to HA-tagged CK1α full-length (FL) or lacking residues 283-337 (ΔC) in HEK293T cells. h, Myc-Venus (M/V)-tagged CARMA1 association with HA-CK1α mutants in HEK293T cells by IP/IB. i, Mapping of the minimal CK1α-binding domain of CARMA1 by IP/IB in HEK293T cells expressing HA-CK1α and M/V-CARMA1 truncation mutants.

Figure 2. Requirement of CK1α for NF-κB activation and proliferation in lymphocytes

a and b, Human peripheral blood T lymphocytes were transfected with siRNA for CK1α, NF-κB p65, BCL10, or scrambled nonspecific (NS) siRNA. IL-2 secretion (mean ± s.d. of triplicate measurements), and CD25 induction 12 hours post-stimulation. CFSE dilution was assessed after 96 hours. Percentage of CD25-positive cells, and of dividing cells is shown. c, NF-κB luciferase assay (mean ± s.d. of triplicate experiments) of Jurkat cells transfected with CK1α (CK1α.2 and CK1α.3), BCL10 or NS siRNA, and stimulated with 1 μg/ml anti-CD3 and anti-CD28, or with 25 ng/ml TNF-α. Left panel, IB for CK1α, BCL10, and β-actin. RLU, relative light units. d, NF-κB luciferase assay of Jurkat stably expressing CK1α-shRNA (sh14 and sh18) reconstituted with a control vector or with mouse CK1α (mCK1α), stimulated with PMA and 100 ng/ml ionomycin, and analyzed as in (c). e and f, IB of control and CK1α-shRNA (sh18) expressing Jurkat exposed to 10 ng/ml PMA and 100 ng/ml ionomycin. g, IP/IB of NS- and CK1α-silenced Jurkat stimulated with 1 μg/ml anti-CD3 and anti-CD28 for 20 min. Arrowhead, CARMA1; black and open squares, IKKβ and phosphorylated-IKKβ.

Figure 3. CK1α kinase activity participates to the negative feedback control of the CBM and NF-κB activation

a, NF-κB luciferase assay (mean ± s.d. of triplicate experiments) of nonspecific (NS)-and CK1α-silenced Jurkat cells reconstituted with an empty vector (−), WT- or D136N-mouse CK1α (mCK1α). RLU, relative luciferase units. b, IB of Jurkat cells expressing WT-, or D136N-mCK1α stimulated with 1 μg/ml anti-CD3 and anti-CD28. c, IB of HEK293T cells expressing Myc/Venus (M/V)-CARMA1 together with an empty vector (−), WT-, or D136N-mCK1α. Filled and open triangles, CARMA1 and its shifted form; Hsp90, loading control. d, Schematic of CARMA1 coiled-coil linker region (CARMA1-CCL, residues 104-660). Red circles, serine/threonine residues. IB of HEK293T cells overexpressing a vector alone (−), WT-, and D136N-CK1α with M/V-CARMA1-CCL(104-660). Open and solid arrowheads, phosphorylated and dephosphorylated CCL. e, Experiment as in (d) using M/V-tagged CCL residues 104-610 or 104-600. Diaphanous (mDIA1), loading control. f, Experiment as in (d) using Myc/Cerulean (M/Cer)-tagged CCL(104-610) with the indicated serine to alanine substitution. g, NF-κB luciferase assay (mean ± s.d. of triplicate experiments) of CARMA1-deficient Jurkat JPM50.6 cells reconstituted with either an empty vector (EV), or the indicated CARMA1 plasmid, and stimulated with PMA and 1 μg/ml anti-CD28.

Figure 4. Role of CK1α in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) survival and NF-κB signaling

a, A genetic screen using 1,854 shRNA vectors targeting 683 genes identified two CK1α shRNAs that block the survival of ABC but not GCB DLBCL cell lines. Shown are the relative fluorescent signals from bar code microarrays comparing shRNA-induced versus uninduced cells 21 days post-shRNA induction. Data are mean ± s.d. of four independent infections of the shRNA retroviral library. b, Survival analysis by flow cytometry of the indicated lymphoma and multiple myeloma cell lines retrovirally infected to express CK1α shRNA and GFP. c, An OCI-Ly3 cell line stably expressing an IκBα-luciferase reporter was retrovirally infected with the indicated shRNAs. Shown is the percentage of luciferase activity in shRNA-induced cells compared to uninduced cells. d, IP/IB of cell lysates from the indicated cell lines. Ub, ubiquitin. e, Immunofluorescent staining for CARMA1 mutant 3 (HA epitope), and endogenous CK1α in OCI-Ly19 retrovirally transduced to express HA-tagged CARMA1 mutant 3. Shown are 2 adjacent cells with nuclei counterstained (blue). f, Indicated ABC DLBCL cells were transduced with WT-CK1α or ΔC-CK1α (Δ283-337), and subsequently with vectors co-expressing GFP and either CK1α shRNA or CARMA1 shRNA. GFP⁺ cell fraction was monitored as in (b).